Preparation of further diagnostic agents

ABSTRACT

Microspheres are prepared by a process comprising (i) spray-drying a solution or dispersion of a wall-forming material in order to obtain intermediate microspheres and (ii) reducing the water-solubility of at least the outside of the intermediate microspheres. Suitable wall-forming materials include proteins such as albumin and gelatin. The microspheres have walls of 40-500 nm thick and are useful in ultrasonic imaging. The control of size, size distribution and degree of insolubilisation and cross-linking of the wall-forming material allows novel microsphere preparations to be produced. In particular, the microspheres may be 15-20 μm, targeted to selected areas of the body or of prolonged life in the circulation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/390,467, filed Sep. 3, 1999, (U.S. Pat. No. 6,416,741), which is acontinuation of U.S. application Ser. No. 08/465,621, filed Jun. 5, 1995(U.S. Pat. No. 6,015,546), which is a continuation of U.S. applicationSer. No. 08/411,815, filed Jun. 28, 1995 (U.S. Pat. No. 6,344,182),which is the National Stage of International Application No.PCT/GB93/02091, filed Oct. 8, 1993.

The present invention relates to the preparation of diagnostic agentscomprising hollow microcapsules used to enhance ultrasound imaging.

The fact that air bubbles in the body can be used for echocardiographyhas been known for some time. Bubble-containing liquids can be injectedinto the bloodstream for this purpose (see Ophir et al (1980)“Ultrasonic Imaging” 2, 67-77, who stabilised bubbles in a collagenmembrane, U.S. Pat. No. 4,446,442 (Schering) and EP-A-131 540(Schering)) and U.S. Pat. Nos. 4,718,433, 4,774,958 and 4,844,882disclose the use of bubbles prepared by sonicating an albumin solution.However, the size distribution of the bubbles is apparentlyuncontrollable and the bubbles disappear when subjected to pressureexperienced in the left ventricle (Shapiro et al (1990) J. Am. Coll.Cardiology, 16(7), 1603-1607).

EP-A-52575 discloses, for the same purpose, solid particles which havegas entrained in them, the gas being released from the particles in thebloodstream.

EP 458 745 (Sintetica) discloses a process of preparing air- orgas-filled microballoons by interfacial polymerisation of syntheticpolymers such as polylactides and polyglycolides. WO 91/12823 (DeltaBiotechnology) discloses a similar process using albumin. Wheatley et al(1990) Biomaterials 11, 713-717 discloses ionotropic gelation ofalginate to form microbubbles of over 30 μm diameter. WO 91/09629discloses liposomes for use as ultrasound contrast agents. Ourco-pending patent application PCT/GB92/00643 (published since thepriority date of this application as WO 92/18164) discloses aspray-drying method which leads to particularly advantageousmicrospheres having the required strength and tightly controlled sizedistribution. Other spray-drying processes, for different purposes, weredisclosed in Przyborowski et al (1982 Eur. J. Nucl. Med. 7, 71-72),namely the preparation of human serum albumin (HSA) microspheres forradiolabelling and subsequent use in scintigraphic imaging of the lung.

The Przyborowski et al article refers to two earlier disclosures ofmethods of obtaining albumin particles for lung scintigraphy. Aldrich &Johnston (1974) Int. J. Appl. Rad. Isot. 25, 15-18 disclosed the use ofa spinning disc to generate 3-70 μm diameter particles which are thendenatured in hot oil. The oil is removed and the particles labelled withradioisotopes. Raju et al (1978) Isotopenpraxis 14(2), 57-61 used thesame spinning disc technique but denatured the albumin by simply heatingthe particles. In neither case were hollow microspheres mentioned andthe particles prepared were not suitable for echocardiography.

We have now developed our previous spray-drying process (WO 92/18164)and adapted it to produce further advantageous products.

One aspect of the present invention provides a process comprising afirst step of atomising a solution or dispersion of a wall-formingmaterial in order to obtain (i) hollow microspheres of 15-20 μmdiameter, (ii) hollow microspheres having a prolonged half-life in thehuman bloodstream or (iii) hollow microspheres which are adapted forselective targeting to an area of the human or animal body.

These three microsphere products will be termed herein “the largemicrospheres”, “the long life microspheres” and “the targetedmicrospheres”, respectively.

Preferably, the product obtained in the said process is subjected to asecond step of reducing the water-solubility of at least the outside ofthe said microspheres.

The said two steps may be carried out as a single process or theintermediate product of the first step may be collected and separatelytreated in the second step. These two possibilities are referred tohereinafter as the one step and two step processes.

The wall-forming material and process conditions should be so chosenthat the product is sufficiently non-toxic and non-immunogenic in theconditions of use, which will clearly depend on the dose administeredand duration of treatment. The wall-forming material may be a starchderivative, a synthetic polymer such as tert-butyloxycarbonylmethylpolyglutamate (U.S. Pat. No. 4,888,398) or a polysaccharide such aspolydextrose or starch.

Generally, the wall-forming material can be selected from mosthydrophilic, biodegradable physiologically compatible polymers. Amongsuch polymers one can cite polysaccharides of low water solubility,polylactides and polyglycolides and their copolymers, copolymers oflactides and lactones such as ε-caprolactone, δ-valerolactone,polypeptides, and proteins such as gelatin, collagen, globulins andalbumins. Other suitable polymers include poly(ortho)esters (see forinstance U.S. Pat. Nos. 4,093,709; 4,131,648; 4,138,344; 4,180,646;polylactic and polyglycolic acid and their copolymers, for instanceDEXON (see J. Heller (1980) Biomaterials 1, 51;poly(DL-lactide-co-δ-caprolactone), poly(DL-lactide-co-δ-valerolactone),poly(DL-lactide-co-g-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones(Polymer 23 (1982), 1693); polyphosphazenes (Science 193 (1976), 1214);and polyanhydrides. References on biodegradable polymers can be found inR. Langer et al (1983) Macromol. Chem. Phys. C23, 61-125.Polyamino-acids such as polyglutamic and polyaspartic acids can also beused as well as their derivatives, ie partial esters with lower alcoholsor glycols. One useful example of such polymers ispoly-(t,butyl-glutamate). Copolymers with other amino-acids such asmethionine, leucine, valine, proline, glycine, alamine, etc are alsopossible. Recently some novel derivatives of polyglutamic andpolyaspartic acid with controlled biodegradability have been reported(see WO 87/03891; U.S. Pat. No. 4,888,398 and EP 130 935 incorporatedhere by reference). These polymers (and copolymers with otheramino-acids) have formulae of the following type:—(NH—CHA—CO)_(x)(NH—CHX—CO)_(y)

where X designates the side chain of an amino-acid residue and A is agroup of formula —(CH₂)_(n)COOR¹R²OCOR(II), with R¹ and R² being H orlower alkyls, and R being alkyl or aryl; or R and R¹ are connectedtogether by a substituted or unsubstituted linking member to provide 5-or 6-membered rings.

A can also represent groups of formulae:—(CH₂)_(n)COO—CHR¹COOR  (I)and—(CH₂)_(n)CO(NH—CHX—CO)_(m)NH—CH(COOH)—(CH₂)_(p)COOH  (III)and corresponding anhydrides. In all these formulae n, m and p are lowerintegers (not exceeding 5) and x and y are also integers selected forhaving molecular weights not below 5000.

The aforementioned polymers are suitable for making the microspheresaccording to the invention and, depending on the nature of substituentsR, R¹, R² and X, the properties of the wall can be controlled, forinstance, strength, elasticity and biodegradability. For instance X canbe methyl (alanine), isopropyl (valine), isobutyl (leucine andisoleucine) or benzyl (phenylalanine).

Preferably, the wall-forming material is proteinaceous. For example, itmay be collagen, gelatin or (serum) albumin, in each case preferably ofhuman origin (ie derived from humans or corresponding in structure tothe human protein). Most preferably, it is human serum albumin (HA)derived from blood donations or from the fermentation of microorganisms(including cell lines) which have been transformed or transfected toexpress HA.

Techniques for expressing HA (which term includes analogues andfragments of human albumin, for example those of EP-A-322094, andpolymers of monomeric albumin) are disclosed in, for example,EP-A-201239 and EP-A-286424. All references are included herein byreference. “Analogues and fragments” of HA include all polypeptides (i)which are capable of forming a microsphere in the process of theinvention and (ii) of which a continuous region of at least 50%(preferably at least 75%, 80%, 90% or 95%) of the amino acid sequencehas at least 80% sequence identity (preferably at least 90%, 95% or 99%identity) with a continuous region of at least 50% (preferably 75%, 80%,90% or 95%) of human albumin. HA which is produced by recombinant DNAtechniques is particularly preferred. Thus, the HA may be produced byexpressing an HA-encoding nucleotide sequence in yeast or in anothermicroorganism and purifying the product, as is known in the art.

In the following description of preferred embodiments, the term“protein” is used since this is what we prefer but it is to beunderstood that other biocompatible wall-forming materials can be used,as discussed above.

The protein solution or dispersion is preferably 0.1 to 50% w/v, morepreferably about 5.0-25.0% protein, particularly when the protein isalbumin. About 20% is optimal. Mixtures of wall-forming materials may beused, in which case the percentages in the last two sentences refer tothe total content of wall-forming material.

The preparation to be sprayed may contain substances other than thewall-forming material and solvent or carrier liquid. Thus, the aqueousphase may contain 1-20% by weight of water-soluble hydrophilic compoundslike sugars and polymers as stabilizers, eg polyvinyl alcohol (PVA),polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin,polyglutamic acid and polysaccharides such as starch, dextran, agar,xanthan and the like. Similar aqueous phases can be used as the carrierliquid in which the final microsphere product is suspended before use.Emulsifiers may be used (0.1-5% by weight) including mostphysiologically acceptable emulsifiers, for instance egg lecithin orsoya bean lecithin, or synthetic lecithins such as saturated syntheticlecithins, for example, dimyristoyl phosphatidyl choline, dipalmitoylphosphatidyl choline or distearoyl phosphatidyl choline or unsaturatedsynthetic lecithins, such as dioleyl phosphatidyl choline or dilinoleylphosphatidyl choline. Emulsifiers also include surfactants such as freefatty acids, esters of fatty acids with polyoxyalkylene compounds likepolyoxypropylene glycol and polyoxyethylene glycol; ethers of fattyalcohols with polyoxyalkylene glycols; esters of fatty acids withpolyoxyalkylated sorbitan; soaps; glycerol-polyalkylene stearate;glycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols; polyethoxylated soya-oil and castor oil as well ashydrogenated derivatives; ethers and esters of sucrose or othercarbohydrates with fatty acids, fatty alcohols, these being optionallypolyoxyalkylated; mono-, di- and triglycerides of saturated orunsaturated fatty acids, glycerides or soya-oil and sucrose.

Additives can be incorporated into the wall of the microspheres tomodify the physical properties such as dispersibility, elasticity andwater permeability.

Among the useful additives, one may cite compounds which can“hydrophobize” the wall in order to decrease water permeability, such asfats, waxes and high molecular-weight hydrocarbons. Additives whichimprove dispersibility of the microspheres in the injectableliquid-carrier are amphipathic compounds like the phospholipids; theyalso increase water permeability and rate of biodegradability.

Additives which increase wall elasticity are the plasticizers likeisopropyl myristate and the like. Also, very useful additives areconstituted by polymers akin to that of the wall itself but withrelatively low molecular weight. For instance when using copolymers ofpolylactic/polyglycolic type as the wall-forming material, theproperties of the wall can be modified advantageously (enhanced softnessand biodegradability) by incorporating, as additives, low molecularweight (1000 to 15,000 Dalton) polyglycolides or polylactides. Alsopolyethylene glycol of moderate to low MW (eg PEG 2000) is a usefulsoftening additive.

The quantity of additives to be incorporated in the wall is extremelyvariable and depends on the needs. In some cases no additive is used atall; in other cases amounts of additives which may reach about 20% byweight of the wall are possible.

The protein solution or dispersion (preferably solution), referred tohereinafter as the “protein preparation”, is atomised and spray-dried byany suitable technique which results in discrete microspheres of1.00-50.0 μm diameter. These figures refer to at least 90% of thepopulation of microspheres, the diameter being measured with a CoulterMaster Sizer II. The term “microspheres” means hollow particlesenclosing a space, which space is filled with a gas or vapour but notwith any solid materials. Honeycombed particles resembling theconfectionery sold in the UK as “Maltesers” (Regd TM) are not formed. Itis not necessary for the space to be totally enclosed (although this ispreferred) and it is not necessary for the microspheres to be preciselyspherical, although they are generally spherical. If the microspheresare not spherical, then the diameters referred to above relate to thediameter of a corresponding spherical microsphere having the same massand enclosing the same volume of hollow space as the non-sphericalmicrosphere.

The atomising comprises forming an aerosol of the protein preparationby, for example, forcing the preparation through at least one orificeunder pressure into, or by using a centrifugal atomizer in, a chamber ofwarm air or other inert gas. The chamber should ideally be big enoughfor the largest ejected drops not to strike the walls before drying. Thegas or vapour in the chamber is clean (ie preferably sterile andpyrogen-free) and non-toxic when administered into the bloodstream inthe amounts concomitant with administration of the microspheres inechocardiography. The rate of evaporation of the liquid from the proteinpreparation should be sufficiently high to form hollow microspheres butnot so high as to burst the microspheres. The rate of evaporation may becontrolled by varying the gas flow rate, concentration of protein in theprotein preparation, nature of liquid carrier, feed rate of the solutionand, most importantly, the temperature of the gas encountered by theaerosol. With an albumin concentration of 15-25% in water, an inlet gastemperature of at least about 100° C., preferably at least 110° C., isgenerally sufficient to ensure hollowness and the temperature may be ashigh as 250° C. without the capsules bursting. About 180-240° C.,preferably about 210-230° C. and most preferably about 220° C., isoptimal, at least for albumin. The temperature may, in the one stepversion of the process of the invention, be sufficient to insolubiliseat least part (usually the outside) of the wall-forming material andfrequently substantially all of the wall-forming material. Since thetemperature of the gas encountered by the aerosol will depend also onthe rate at which the aerosol is delivered and on the liquid content ofthe protein preparation, the outlet temperature may be monitored toensure an adequate temperature in the chamber. An outlet temperature of40-150° C. has been found to be suitable. Apart from this factor,however, controlling the flow rate has not been found to be as useful ascontrolling the other parameters.

In the two step process, the intermediate microspheres comprisetypically 96-98% monomeric HA and have a limited in vivo life time forultrasound imaging. They may, however, be used for ultrasound imaging(at least in some uses of the microspheres of the invention), or theymay be stored and transported before the second step of the two stepprocess is carried out. They therefore form a further aspect of theinvention.

In the second step of the process, the intermediate microspheresprepared in the first step are fixed and rendered less water-soluble sothat they persist for longer whilst not being so insoluble and inertthat they are not biodegradable. This step also strengthens themicrospheres so that they are better able to withstand the rigours ofadministration, vascular shear and ventricular pressure. If themicrospheres burst, they become less echogenic. Schneider et al (1992)Invest. Radiol. 27, 134-139 showed that prior art sonicated albuminmicrobubbles do not have this strength and rapidly lose theirechogenicity when subjected to pressures typical of the left ventricle.The second step of the process may employ heat (for example microwaveheat, radiant heat or hot air, for example in a conventional oven),ionising irradiation (with, for example, a 10.0-100.0 kGy dose of gammarays) or chemical cross-linking using, for example, formaldehyde,glutaraldehyde, ethylene oxide or other agents for cross-linkingproteins and is preferably carried out on the substantially dryintermediate microspheres formed in the first step, or on a suspensionof such microspheres in a liquid in which the microspheres areinsoluble, for example a suitable solvent. In the one step version ofthe process, a cross-linking agent such as glutaraldehyde may be sprayedinto the spray-drying chamber or may be introduced into the proteinpreparation just upstream of the spraying means. Alternatively, thetemperature in the chamber may be high enough to insolubilise themicrospheres.

The “long life microspheres” and the “targeted microspheres” may, if onewishes, consist of microspheres having a diameter of 0.05 to 50.0 μm(measured in the same way as the intermediate microspheres), but rangesof 0.1 to 20.0 μm and especially 1.0 to 8.0 μm are obtainable with theprocess of the invention and are preferred for echocardiography. We havefound that a range of about 0.5 to 3.0 μm may be especially suitable forthe production of a low contrast image and for use in colour Dopplerimaging, whereas a range of about 4.0 to 6.0 μm may be better for theproduction of sharp images. One needs to take into account the fact thatthe second step may alter the size of the microspheres in determiningthe size produced in the first step.

It has been found that the process of the invention can be controlled inorder to obtain microspheres with desired characteristics. Thus, thepressure at which the protein solution is supplied to the spray nozzlemay be varied, for example from 1.0-10.0×10⁵ Pa, preferably 2.0-6.0×10⁵Pa and most preferably about 5×10⁵ Pa. Other parameters may be varied asdisclosed above and below. In this way, novel microspheres may beobtained.

A further aspect of the invention provides large, long life or targetedhollow microspheres in which more than 30%, preferably more than 40%,50%, or 60%, of the microspheres have a diameter within a 2 μm rangeand, in the case of the long life or targeted microspheres, at least90%, preferably at least 95% or 99%, have a diameter within the range1.0-8.0 μm. In the case of the large microspheres, the correspondingdiameter range is 12-25 μm.

Thus, the interquartile range may be 2 μm, with a median diameter (forthe long life or targeted microspheres) of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0or 6.5 μm.

Thus, at least 30%, 40%, 50% or 60% of the long life or targetedmicrospheres may have diameters within the range 1.5-3.5 μm, 2.0-4.0 μm,3.0-5.0 μm, 4.0-6.0 μm, 5.0-7.0 μm or 6.0-8.0 μm. Preferably a saidpercentage of the said microspheres have diameters within a 1.0 μmrange, such as 1.5-2.5 μm, 2.0-3.0 μm, 3.0-4.0 μm, 4.0-5.0 μm, 5.0-6.0μm, 6.0-7.0 μm or 7.0-8.0 μm.

A further aspect of the invention provides large, long life or targetedhollow microspheres with proteinaceous walls in which at least 90%,preferably at least 95% or 99%, of the microspheres have a diameter inthe range 1.0-8.0 μm (or, in the case of the large microspheres, 12-25μm); at least 90%, preferably at least 95% or 99%, of the microsphereshave a wall thickness of 40-500 nm, preferably 100-500 nm; and at least50% of the protein in the walls of the microspheres is cross-linked.

Scanning electron microscopy of the microcapsules shows that they arehollow spheres with no solid matter other than in the wall. Hence, thewall thickness can either be measured microscopically or can becalculated as follows. The mass of wall-forming material in each of thesprayed droplets is given by $\begin{matrix}\begin{matrix}{{Mass} = {\left( {{volume}\quad{of}\quad{droplet}} \right) \times \left( {{concentration}\quad{of}\quad{wall}\text{-}{forming}}\quad \right.}} \\{\left. {{material}\quad{in}\quad{solution}\quad{sprayed}} \right)\quad} \\{= {\frac{4}{3}\pi\quad r_{e}^{3}c}}\end{matrix} & (I)\end{matrix}$

where r_(e) is the radius of the droplet and c is the saidconcentration.

Our studies have shown that the external dimension of the droplet isessentially unchanged whilst the solvent is evaporated off. The mass ofwall-forming material in the dried microcapsule is therefore given by$\begin{matrix}{{mass} = {\frac{4}{3}{\pi\left( {r_{e}^{3} - r_{i}^{3}} \right)}\rho}} & ({II})\end{matrix}$

where r_(e) is the external radius of the microcapsule (same as that ofthe droplet), r_(i) is the internal radius of the microcapsule and ρ isthe density of the wall-forming material. The wall thickness is thenrepresented by r_(e)−r_(i). The quantity r_(e) is known fromstraightforward measurement of the microcapsules using a CoulterCounter, and r_(i) is obtained by $\begin{matrix}{r_{i} = \sqrt[3]{r_{e}^{3} - \frac{r_{e}^{3}c}{\rho}}} & ({III})\end{matrix}$

Hence, for an external diameter of 5 μm (external radius of 2.5 μm), aconcentration in the solution sprayed of 0.2 g/ml (20%) and a walldensity of 1.31 g/cm³ (determinable by helium pycnometry), the wallthickness can be calculated to be 134 nm.

Preferably, at least 75%, 90%, 95%, 98.0%, 98.5% or 99% of the proteinin any of the three kinds of microspheres of the invention issufficiently cross-linked to be resistant to extraction with a 1% HClsolution for 2 minutes. Extracted protein is detected using theCoomassie Blue protein assay, Bradford. The protein content in thewashings is expressed as a percentage of the original mass ofmicrocapsules.

The degree of cross-linking is controlled by varying the heating,irradiation or chemical treatment of the protein. During thecross-linking process, protein monomer is cross-linked and quicklybecomes unavailable in a simple dissolution process, as detected by gelpermeation HPLC or gel electrophoresis, as is shown in Example 8 below.Continued treatment leads to further cross-linking of alreadycross-linked material such that it becomes unavailable in the HClextraction described above. During heating at 175° C., rHA microspheresin accordance with the invention lose about 99% of HCl-extractableprotein over the course of 20 minutes, whereas, at 150° C., 20 minutes'heating removes only about 5% HCl-extractable protein, 30 mins removes47.5%, 40 mins 83%, 60 mins 93%, 80 mins 97% and 100 mins removes 97.8%of the HCl-extractable protein. To achieve good levels of cross-linkingtherefore, the microspheres may be heated at 175° C. for at least 17(preferably 20-40 mins, most preferably 35-40 mins) mins, at 150° C. forat least 80 mins and at other temperatures for correspondingly longer orshorter times. We have found that serum-derived albumin needs less timeto cross-link than rHA.

The injectable microspheres of the present invention can be stored dryin the presence or in the absence of additives to improve conservationand prevent coalescence. As additives, one may select from 0.1 to 25% byweight of water-soluble physiologically acceptable compounds such asmannitol, galactose, lactose or sucrose or hydrophilic polymers likedextran, xanthan, agar, starch, PVP, polyglutamic acid, polyvinylalcohol(PVA) and gelatin.

In order to minimise any agglomeration of the microspheres, themicrospheres can be milled with a suitable inert excipient using aFritsch centrifugal pin mill equipped with a 0.5 mm screen, or a GlenCreston air impact jet mill. Suitable excipients are finely milledpowders which are inert and suitable for intravenous use, such aslactose, glucose, mannitol, sorbitol, galactose, maltose or sodiumchloride. Once milled, the microspheres/excipient mixture can besuspended in aqueous medium to facilitate removal ofnon-functional/defective microspheres. Upon reconstitution in theaqueous phase, it is desirable to include a trace amount of surfactantto prevent agglomeration. Anionic, cationic and non-ionic surfactantssuitable for this purpose include poloxamers, sorbitan esters,polysorbates and lecithin.

The microsphere suspension may then be allowed to float, or may becentrifuged to sediment any defective particles which have surfacedefects which would, in use, cause them to fill with liquid and be nolonger echogenic.

The microsphere suspension may then be remixed to ensure even particledistribution, washed and reconstituted in a buffer suitable forintravenous injection such as 0.15M NaCl 0.01 mM Tris pH 7.0. Thesuspension may be aliquoted for freeze drying and subsequentsterilisation by, for example, gamma irradiation, dry heating orethylene oxide.

An alternative method for deagglomeration of the insolubilised or fixedmicrospheres is to suspend them directly in an aqueous medium containinga surfactant chosen from poloxamers, sorbitan esters, polysorbates andlecithin. Deagglomeration may then be achieved using a suitablehomogeniser.

The microsphere suspension may then be allowed to float or may becentrifuged to sediment the defective particles, as above, and furthertreated as above.

Although the microspheres of this invention can be marketed in the drystate, more particularly when they are designed with a limited life timeafter injection, it may be desirable to also sell ready-madepreparations, ie suspensions of microspheres in an aqueous liquidcarrier ready for injection.

The product is generally, however, supplied and stored as a dry powderand is suspended in a suitable sterile, non-pyrogenic liquid just beforeadministration.

A further aspect of the invention provides large, long life or targetedhollow microspheres, at least 10% of the microspheres, when suspended inwater, being capable of surviving a 0.25 s application of a pressure of2.66×10⁴ Pa without bursting, collapsing or filling with water. Thetransient maximum pressure in the human left ventricle is about 200 mmHg(2.66×10⁴ Pa). Preferably 50%, 75%, 90% or 100% survive the said 0.25 sapplication of 2.66×10⁴ Pa when tested as above, ie remain echogenic. Invivo, preferably the same percentages will remain echogenic during onepassage through both ventricles of the heart.

The “large” microspheres of the invention are characterised by the factthat at least 90%, preferably at least 95% or 99%, of the microsphereshave a diameter within the range 10.1-19.9 μm, preferably 13-18 μm.

It should be noted that these microspheres are “large” only in relationto the preferred microspheres of our earlier patent application WO92/18164 and in relation to the preferred sizes of long life andtargeted microspheres disclosed herein; prior art microspheres werefrequently larger than 25 μm.

The large microspheres of the invention may be produced by controllingthe parameters of the spray-drying process. The concentration of thewall-forming material in the liquid to be sprayed may be the same as forthe smaller microspheres described above, namely 0.1-50.0% w/v(preferably about 5.0-25.0%, especially when the wall-forming materialis albumin), as may the temperature in the warm chamber (100-250° C.,preferably 200-250° C.) and the second step of the process, but thespraying pressure is reduced to less than 2 bar (2×10⁵ Pa) and ispreferably no more than 1.8×10⁵ Pa, 1.5×10⁵ Pa or 1.3×10⁵ Pa. A minimumpressure of 1×10⁵ Pa is preferred.

The large microspheres of the invention are suitable for use as adeposit echocontrast agent to delineate under-perfused areas ofmicrocirculation. We have found that microspheres of mean size 15.0 μmhave echogenicities some 4.6×10⁴ fold higher than similar microspheresof mean size 5.0 μm. Hence, a relatively low dose can be used to imageregions deep inside the body which are inaccessible to normal ultrasoundtechniques. The microspheres can be delivered by known techniques usinga catheter to deliver the microspheres to, for example, the capillariesof the liver, kidney or coronary blood vessels. An advantage, comparedto classical radiolabelled microsphere studies, is that, followingarterial administration, catheter withdrawal and patient stabilisation,multiple plane images may be taken to build a 3D perfusion map of themyocardium or similar capillary bed. Regional myocardial blood flow canbe qualitatively assessed in patients with coronary artery disease atthe time of angiography by imaging the heart following the directintracoronary injection of the microspheres. These microspheres aretrapped in the microvasculature of the heart during the initial transmitthrough the coronary circulation. Since only a very small fraction ofthe capillaries or arterioles is embolized, no detectable adversehaemodynamic or electrophysiological effects are expected. When nutrientblood flow to a segment of the left ventricular myocardium isdiminished, as in a region of myocardial scar or in a region supplied byan occluded or severely stenotic coronary artery, the number ofmicrospheres delivered to these segments is reduced. This is appreciatedas a focal reduction in activity secondary to regional underperfusion.Because the microspheres are introduced into the arteries, removal ofthe microspheres in the capillaries of the lung is avoided.

In the context of angiography, a catheter is placed within the leftventricle via insertion in the femoral artery. X-ray opaque dyes areinjected both in the left ventricle and within the coronary arteriesthemselves. Injection of such agents enables the visualisation ofvessels to the 100 μm diameter level by projecting the 3D informationonto a 2D plane. Currently angiography enables stenosis of the majorcoronary arteries to be identified.

The use of the large microspheres of the invention with ultrasoundtechnology may enable the generation of multiple tomographic images andalso 3D reconstruction of images. With the microspheres depositing forsufficient time to enable tomographic images or 3D image reconstructionof the vascular bed, perfusion beds may be delineated. Therefore, as anadjunct to angiography to identify the major causative lesion, a depositechocontrast agent constituted by the large microspheres of theinvention may enable 3D perfusion territories to be identified.

Due to the pressure stability of the preferred microspheres, they retainair and hence echogenicity for a substantial period of time. Themicrospheres may deposit in the vasculature following catheteradministration in a manner similar to classical microsphere studies,reflecting the amount of flow to any given perfusion territory. Imagingof the territory may then be made after catheter withdrawal and patientstabilisation, to enable more optimal images in multiple planes to begathered. Comparison with a baseline unenhanced image thus enables theperfusion, following a corrective procedure, to be assessed.

The microspheres may be tailored for intracoronary use not only bymanipulation of their size and pressure stability but also by their rateof biodegradation.

For intracoronary use, it is preferable to crosslink the large (10-20μm) microcapsules at 175° C. for a period of 18-60 minutes, morepreferably 20-40 minutes and most preferably 35-40 minutes. This yieldsmicrocapsules that are pressure resistant but have a shortened tissuehalf life compared to the microcapsules of WO 92/18164 and therefore aremore applicable to use in the microcirculation of the myocardium. Thetissue half-life can be measured by labelling the microcapsules with¹²⁵I by the Chloramine T method and assessing the organ content ofmicrocapsules by necropsy or the release of ¹²⁵I into the urine andfaeces.

The “targeted” microspheres of the invention are characterised by havingin or on their walls a material to direct or target the microspheres toa desired location in the body.

The “targeted” microspheres of the invention may be prepared byincluding in or on the wall of the microsphere material which alters theelectrical charge of the microsphere.

Thus, a positive or negative charge can be imparted by applying apositively or negatively charged material, respectively, or existingpositive or negative charges can be reduced or eliminated. These effectscan be achieved in a variety of ways. The final product (ie pressresistant) microspheres produced by the basic one or two step processdescribed above may be milled as described above and resuspended at amicrosphere concentration of 1.0-250×10⁶/ml in: a 0.5-20.0% w/v solution(preferably 1.0-10.0% w/v, for example about 5%) of a positively ornegatively charged material (if polymeric of 1-30 kD, preferably 5-15kD) and incubated for 5-60 hours (preferably about 8-24 hours) at 5-30°C. (preferably about 20° C.). Positively charged polyamino acids includepolylysine, polyaspartamide, polyarginate and polyhistidine. Negativelycharged polyamino acids include polyglutamate and polyaspartate. Othernegatively charged polymers include phospholipids, hyaluronic acid andpolygluconic acid. An advantage of such coated echocontrast agents is toincrease the echogenicity of the blood pool to enable signal enhancementof doppler signals.

Alternatively, and more preferably, positive or negative charges onmicrospheres may be increased by incorporating the material in thespraydrying feedstock in the range of 1-30%, preferably 2-10% w/v. Thislatter method is particularly preferred for polyglutamate, and fornegatively charged additives generally.

Other materials which can be used in the same way to impart a negativecharge include anhydrides and chlorides of C₁₋₁₀ organic acids, such asacetic, fumaric and succinic acids. A final concentration of thechloride or anhydride of 5-1000 mg/ml is generally suitable, in anon-polar solvent such as dimethylformamide or tetrahydrofuran. Anincubation time of 0.5-5 hours, preferably about 1 hour, at 5-30° C.,preferably about 20° C., is suitable, followed by washing with excesswater.

Existing negative charges on the microspheres prepared by the basicspray-drying process may be removed by exposing the microspheres to acarbodiimide agent such asN-ethyl-N¹-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), at aconcentration of about 5-1000 mg/ml for a period of about 5-30 hours(preferably about 16 hours) at 5-30° C. (preferably about 20° C.).Excess reagent is then quenched with, for example, ethanolamine to anequivalent concentration during a further such incubation before themicrospheres are washed.

The electrophoretic mobility of the microspheres may be assessed in aMalvern Zeta sizer or in a Pen Kem System 3000 (USA) minielectrophoresiscell, for example for 20 particles in buffers of pH 4-10. Preferably,the electrophoretic mobility is in one of the ranges plus or minus0.001-5.0×10⁻⁸ m/sec/v/cm. In these ranges the charge upon themicrospheres alters their circulatory behaviour. More preferably, themobility is in one of the ranges plus or minus 0.01 to 0.5×10⁻⁸m/sec/v/cm, suitably in one of the ranges plus or minus 0.1 to 0.5×10⁻⁸m/sec/v/cm.

In all of these methods of altering the charge on the microspheres, theresulting microspheres may finally be formulated for storage asdescribed above, for example suspending them in a mannitol/Pluronic F68solution, flash freezing and freeze-drying.

The surface charge of microcapsules can affect the imaging properties ofthe product through its influence on the in vivo fate of particles. Forexample, it is known that after intravenous injection negatively chargedpolystyrene particles are taken up at high efficiency by the liver,whereas particles with a positive charge accumulate initially in thelung. Additionally, it is known that the endothelial cell surface iscoated with a glycocalyx carrying a net negative charge at physiologicalpH values. The inner surface of endothelium may therefore be stainedwith collodial iron particles carrying a net positive charge. Therefore,in areas of slow or sluggish flow, such as that experienced in thecapillary beds of the peripheral vasculature, liver, kidney andmyocardium, increasing the net positive charge on the microcapsule shelland endothelial lining may lead to hindered transit through themicrocirculation. This creates the possibility of extended imagingwindows or even deposit echocontrast agents for analysis of themicrovasculature following intravenous administration.

The “long-life” microspheres have an increased circulation time in thebody, such that serum t_(1/2) is at least 5 minutes, preferably at least10 minutes and most preferably at least 15 minutes. Such increasedcirculation times may be achieved by coating the microspheres with amaterial which directs the microspheres away from thereticul-endothelial system.

In vivo t½ may be assessed by labelling the microcapsules with ¹²⁵Iusing the well known Chloramine T method, and administering them intothe ear vein of a male adult New Zealand rabbit as is generallydescribed in Specific Example 10 below. The serum level of ¹²⁵I ismeasured by gamma counting.

For example, the said material may be one which reduces or substantiallyprevents “opsonization”, the deposition of proteinaceous material (suchas fibrinogen) on the microspheres, thus directing the microspheres awayfrom the liver and spleen. Suitable materials with which to coat themicrospheres include block copolymers of the poloxamer series (iepolyethylene glycol/polyethylene oxide copolymers), such as poloxamer338, poloxamer 407 and poloxamer 908.

By prolonging the circulatory half-life of highly pressure resistantair-containing microcapsules, areas of very low flow such as found inthe capillary beds are detectable beyond enhanced doppler studies.Abnormal blood flow associated with hepatocellular carcinomas, renalcarcinomas, and breast tumours can be detected with use of Dopplertechniques. In general, larger malignant tumours show the greatestsignal changes, and the abnormal Doppler signals become more difficultto detect in smaller tumours. With malignant breast tumours, forinstance, the low signal strength from moving scatterers whose echo is“diluted” by that of stationary solid tissue is one limiting factor inthe detection of small tumours. One criterion for the Doppler detectionof tumour flow is the inhomogeneity of the spatial distribution ofvessels after neovascularization. Contrast enhancement allows thedisplay of smaller vessels and hence increase the utility of thiscriterion in colour Doppler studies. The agent may enhance backscatterin both tumour and normal vessels. Enhanced blood reflectivity improvesdetection and differentiation of small tumours in such organs as thebreast, liver, kidneys, pancreas and ovaries.

Also, the ultrasound contrast agent may help differentiate areas ofnormal vascularity from areas of reduced or absent flow due to thepresence of tumour or necrosis. The demonstration of normal parenchymalarterial flow within areas that were considered abnormal may help todistinguish normal parenchyma from pseudotumours (focal fattyinfiltration of the liver or renal columns of Bertin). Ultrasoundcontrast agents al so may enhance echoes from arterial blood for thedetection of ischemia or occlusion. In cases of partial occlusion, theflow is often fast enough for Doppler detection, but the quantity ofblood (which, with tissue attenuation, determines the signal strength)passing through the narrowing may not be great enough to be detectedwith current Doppler equipment. Under certain circumstances, theintroduction of more reflectors can aid delineation of the site ofnarrowing. A contrast agent may also aid the visualization ofcollaterals caused by occlusion or severe stenosis.

The long-life microspheres are prepared in the same way as the targetedmicrospheres described above, in other words the coating material may beapplied to a suspension of the spray-dried microspheres before they arefreeze-dried or included in the spray feedstock.

A suspension of the microspheres of the invention is generallyadministered by injection of about 1.0-10.0 ml into a suitable vein suchas the cubital vein or other bloodvessel. A microsphere concentration ofabout 1.0×10⁵ to 1.0×10¹² particles/ml is suitable, preferably about5.0×10⁵ to 5.0×10⁹.

Although ultrasonic imaging is applicable to various animal and humanbody organ systems, one of its main applications is in obtaining imagesof myocardial tissue and perfusion or blood flow patterns.

The techniques use ultrasonic scanning equipment consisting of a scannerand imaging apparatus. The equipment produces visual images of apredetermined area, in this case the heart region of a human body.Typically, the transducer is placed directly on the skin over the areato be imaged. The scanner houses various electronic components includingultrasonic transducers. The transducer produces ultrasonic waves whichperform a sector scan of the heart region. The ultrasonic waves arereflected by the various portions of the heart region and are receivedby the receiving transducer and processed in accordance with pulse-echomethods known in the art. After processing, signals are sent to theimaging apparatus (also well known in the art) for viewing.

In the method of the present invention, after the patient is “prepped”and the scanner is in place, the microsphere suspension is injected, forexample through an arm vein. The contrast agent flows through the veinto the right venous side of the heart, through the main pulmonary arteryleading to the lungs, across the lungs, through the capillaries, intothe pulmonary vein and finally into the left atrium and the leftventricular cavity of the heart.

With the microspheres of this invention, observations and diagnoses canbe made with respect to the amount of time required for the blood topass through the lungs, blood flow patterns, the size of the leftatrium, the competence of the mitral valve (which separates the leftatrium and left ventricle), chamber dimensions in the left ventricularcavity and wall motion abnormalities. Upon ejection of the contrastagent from the left ventricle, the competence of the aortic valve alsomay be analyzed, as well as the ejection fraction or percentage ofvolume ejected from the left ventricle. Finally, the contrast patternsin the tissue will indicate which areas, if any, are not beingadequately perfused.

In summary, such a pattern of images will help diagnose unusual bloodflow characteristics within the heart, valvular competence, chambersizes and wall motion, and will provide a potential indicator ofmyocardial perfusion.

The microspheres may permit left heart imaging from intravenousinjections. The albumin microspheres, when injected into a peripheralvein, may be capable of transpulmonary passage. This results inechocardiographic opacification of the left ventricle (LV) cavity aswell as myocardial tissue.

Besides the scanner briefly described above, there exist otherultrasonic scanners, examples of which are disclosed in U.S. Pat. Nos.4,134,554 and 4,315,435, the disclosures of which are hereinincorporated by reference. Basically, these patents relate to varioustechniques including dynamic cross-sectional echography (DCE) forproducing sequential two-dimensional images of cross-sectional slices ofanimal or human anatomy by means of ultrasound energy at a frame ratesufficient to enable dynamic visualisation of moving organs. Types ofapparatus utilised in DCE are generally called DCE scanners and transmitand receive short, sonic pulses in the form of narrow beams or lines.The reflected signals' strength is a function of time, which isconverted to a position using a nominal sound speed, and is displayed ona cathode ray tube or other suitable devices in a manner somewhatanalogous to radar or sonar displays. While DCE can be used to produceimages of many organ systems including the liver, gall bladder, pancreasand kidney, it is frequently used for visualisation of tissue and majorblood vessels of the heart.

The microspheres may be used for imaging a wide variety of areas, evenwhen injected at a peripheral venous site. Those areas include (withoutlimitation): (1) the venous drainage system to the heart; (2) themyocardial tissue and perfusion characteristics during an exercisetreadmill test or the like; and (3) myocardial tissue after an oralingestion or intravenous injection of drugs designed to increase bloodflow to the tissue. Additionally, the microspheres may be useful indelineating changes in the myocardial tissue perfusion due tointerventions such as (1) coronary artery vein grafting; (2) coronaryartery angioplasty (balloon dilation of a narrowed artery); (3) use ofthrombolytic agents (such as streptokinase) to dissolve clots incoronary arteries; or (4) perfusion defects or changes due to a recentheart attack.

Furthermore, at the time of a coronary angiogram (or a digitalsubtraction angiogram) an injection of the microspheres may provide datawith respect to tissue perfusion characteristics that would augment andcomplement the data obtained from the angiogram procedure, whichidentifies only the anatomy of the blood vessels.

Through the use of the microspheres of the present invention, othernon-cardiac organ systems including the liver, spleen and kidney thatare presently imaged by ultrasonic techniques may be suitable forenhancement of such currently obtainable images, and/or the generationof new images showing perfusion and flow characteristics that had notpreviously been susceptible to imaging using prior art ultrasonicimaging techniques.

Preferred aspects of the present invention will now be described by wayof example and with reference to

FIG. 1, which is a partly cut away perspective view from the front andone side of suitable spray-drying apparatus for the first stage of theprocess of the invention,

FIG. 2, which is a graph showing how the degree of fixation of themicrosphere walls (in this case albumin) may be controlled by varyingthe temperature and the heating time in the second step of the process,

FIG. 3, which is a graph showing how the pressure resistivity of themicrospheres may be varied by altering the length of the heating time inthe second step of the process,

FIG. 4 is a graph showing how the in vitro biodegradation rate may bevaried by varying the length of heating time in the second step of theprocess, assessed by a turbidimetric measurement to measuredisappearance of microcapsules, and

FIGS. 5 a and 5 b are respective still copies from video tape showingthe appearance of pig myocardium before and after injection of 4 millionof the large microcapsules of the invention into the left ventricle.

GENERAL PREPARATIVE EXAMPLE 1

A suitable spray dryer (FIG. 1) is available from A/S Niro Atomizer,Soeborg, Denmark under the trade designation “Mobile Minor”. Details ofits construction are given immediately before the claims herein. Itcomprises a centrifugal atomizer (Type M-02/B Minor), driven by an airturbine at an air pressure of min 4 bar and up to max 6 bar. At 6 bar anatomizer wheel speed of approx 33,000 rpm is reached. Turning on and offthe compressed air to the atomizer is done by means of a valve placed inthe instrument panel. The maximum consumption of compressed air to theatomizer is 17 Nm³/h at a pressure of 6 bar. All parts coming intocontact with the liquid feed and powder are made of stainless steel AISI316, except for the pump feed tube and the atomizer wheel, which is madeof stainless steel AISI 329, made to resist high centrifugal force. Thestainless steel interconnecting pipe system 4 can easily be strippeddown for cleaning.

The drying chamber has an inside made of stainless steel AISI 316, wellinsulated with Rockwool, and covered outside with a mild steel sheeting.The drying chamber is provided with a side light and observation panefor inspection during the operation and steps 5 for access to thechamber top. The roof of the drying chamber is made inside of stainlesssteel AISI 316 and outside of stainless steel AISI 304. There is aswitch 6 for an air valve for activation of the pneumatic lifting devicewhen raising the chamber lid.

An air disperser 2 made of stainless steel AISI 304 is used fordistribution of the air in the drying chamber in order to achieve thebest possible drying effect. Swirling air is directed around the vaneddisc atomiser. An air duct, made of stainless steel AISI 316, provideslateral transportation of the exhaust air and the powder to the cyclone7, which is made of stainless steel AISI 316 and designed to separatethe powder and air.

A closing valve of the butterfly valve type, also made of stainlesssteel AISI 316 and having a gasket of silicone rubber, is used forpowder discharge under the cyclone into a powder collecting glass jar 8tightly placed under the cyclone by means of a spring device.

A centrifugal exhaust fan 10 made of silumin, complete with 3-phasesquirrel-cage motor, 0.25 kW, and V-belt drive with belt-guard, drawsair and powder through the drying chamber and cyclone. There is a switch11 for air flow control via a damper.

An air heater 12 heats the drying air by means of electricity (totalconsumption 7.5 kWh/h, infinitely variable) and can give inlet airtemperatures of up to about 350° C., although this is generally too highfor preparing the microspheres of the invention.

The evaporative capacity is as follows:

Evaporative Capacity

Inlet Air Outlet Air Evaporative Drying Air Temperature TemperatureCapacity 85 kg/h 150° C. 80° C. 1.3 kg/h. 85 kg/h 170° C. 85° C. 1.7kg/h 80 kg/h 200° C. 90° C. 2.5 kg/h 80 kg/h 240° C. 90° C. 3.4 kg/h 75kg/h 350° C. 90° C. 7.0 kg/h

Equipment for two-fluid nozzle atomization may be added, which is madeof stainless steel AISI 316, consisting of entrance pipe with nozzleholder and nozzle, to be placed in the ceiling of the drying chamber.The equipment includes an oil/water separator, reduction valve andpressure gauge for compressed air to the two-fluid nozzle. Consumptionof compressed air: 8-15 kg/h at a pressure of 0.5-2.0 bar (0.5-2.0×10⁵Pa).

A suitable feed pump for transport of wall-forming preparation feed froma reservoir 1 to the atomizer nozzle 3 is a peristaltic pump. The pumpis provided with a motor (1×220V, 50 Hz, 0.18 kW) and a continuouslyvariable gear for manual adjustment. A feed pipe made of silicone hoseleads from a feed tank (local supply) 1 through the feed pump to therotary or nozzle atomization device 3.

An absolute air filter, consisting of prefilter, filter body instainless steel and absolute air filter, is used for the treatment ofthe ingoing drying air to render it completely clean. The wholeapparatus is controlled via an instrument panel 9.

A 20% solution of sterile, pyrogen-free rHA in pyrogen-free water(suitable for injection) was pumped to the nozzle of a two fluid nozzleatomiser mounted in the commercial spray drying unit described above.The peristaltic pump speed was maintained at a rate of approximately 10ml/minute such that with an inlet air temperature of 220° C. the outletair temperature was maintained at 95° C.

Compressed air was supplied to the two fluid atomising nozzle at 2.0-6.0Bar (2.0-6.0×10⁵ Pa). In this range microspheres with a mean size of4.25-6.2 μm are obtained.

Typically an increase in mean particle size (by reduced atomisationpressure) led to an increase in the amount of microspheres over 10 μm insize (see Table 1).

TABLE 1 EFFECTS OF ATOMISATION PRESSURE ON FREQUENCY OF MICROSPHERESOVER 10 μM IN DIAMETER Atomisation Pressure (× 10⁵ Pa) % Frequency over10 μm 6.0 0.8 5.0 3.0 3.5 6.6 2.5 8.6 2.0 13.1

In the second step of the process, 5 g of microspheres were heated in aglass beaker using a Gallenkamp fan oven. A temperature of 175° C. for 1hour was sufficient to yield microspheres with 100% fixation asdetermined by HPLC. The effect of this heat fixation was to increase thein vitro echogenic half life from a few seconds to in excess of 30minutes. By altering the temperature and length of incubation it ispossible to vary the degree of fixation between about 5% and 100%.Examples of heat fixation profiles of varying temperatures are shown inFIG. 2.

Following heat fixation, the microspheres were deagglomerated anddispersed into water in one of two ways. Method 1 involved first mixingthe heat fixed spheres with an equal weight of finely milled lactose(mean diameter 5 μm). The mixture was then passed through a Fritschcentrifugal mill with a 0.5 mm screen and 12 tooth rotor. The milledspheres were collected and passed through the mill a second time toensure complete mixing had occurred. The milled powder was thenresuspended in water containing 1 mg·ml⁻¹ Pluronic F68. Typically 10 gof microspheres and lactose was added to 100 ml of water and PluronicF68. Method 2 for deagglomeration involves adding 5 g of the heat-fixedmicrospheres to 100 ml of water containing 100 mg of Pluronic F68. Themicrospheres were dispersed using a Silverson homogeniser (model M4Rwith a 2.54 cm tubular homogenising probe and a high shear screen) andhomogenising for 60 seconds.

The resuspended spheres were separated into intact (gas containing) andbroken spheres using a flotation technique. The gas-containing sphereswere seen to float to the surface over a 1 hour period and were decantedfrom the sinking fraction which does not contain the gas required.

The separation process can be accelerated by centrifugation. A 30 secondcentrifugation at 5000×g is sufficient to separate the two fractions.

Following separation the intact microspheres were freeze-dried in thepresence of lactose and Pluronic F68. Optimal conditions for freezedrying involved resuspending 30 mg of microspheres in 5 ml of watercontaining 50 mg of lactose and 5 mg of Pluronic F68. The freeze-driedmicrospheres can be redispersed in a liquid (eg water, saline) to give amonodisperse distribution.

GENERAL PREPARATIVE EXAMPLE 2

The process of Example 1 was repeated but with the following differencesin the first step: a centrifugal atomiser was used instead of a twofluid nozzle; the inlet temperature was 150° C. (with the outlet airtemperature still being sustained at 105° C.); and compressed air wassupplied to the nozzle at 1.0-6.0×10⁵ Pa. The wheel rotated at 20-40,000rpm and delivered droplets, and subsequently microspheres, with a numbermean diameter in the 1.0-8.0 μm range.

GENERAL PREPARATIVE EXAMPLE 3

The second step of the process of Example 1 or 2 was varied as follows.A small aliquot of the microspheres (0.5 g) was heated in a microwaveoven such that it received 300-350 watt hours of microwave heat at 2500mHz. This yielded microspheres in which 90-95% of the monomeric rHA wasinsoluble (as determined by gel permeation chlomatography) and as aresult of this heat fixation their in vitro echogenic half-lifeincreased from a few seconds to in excess of 30 minutes.

GENERAL PREPARATIVE EXAMPLE 4

The second step of the process of Example 1 or 2 was varied as follows.A small aliquot of the microspheres (0.5 g) was sealed under argon in aglass vial. The vial was cooled to 4° C. and then irradiated with a ⁶⁰Cogamma radiation source to deliver a 15.0 kGy dose of gamma rays. Theirradiation resulted in the formation of microspheres in which 10-15% ofthe monomeric albumin was insoluble.

GENERAL PREPARATIVE EXAMPLE 5

The second step of the process of Example 1 or 2 was varied as follows.A small aliquot of the microspheres (0.5 g) was sealed under argon in aglass vial. The vial was cooled to 4° C. and then irradiated with a ⁶⁰Cogamma radiation source to deliver a 50.0 kGy dose of gamma rays to themicrospheres. Following irradiation, the microspheres were incubated inoxygen at 50° C. for 6 hours. The irradiation resulted in the formationof microspheres in which 50-60% of the monomeric rHA was insoluble.

GENERAL PREPARATIVE EXAMPLE 6

The second step of the process of Example 1 or 2 was varied as follows.

A small aliquot of microspheres (0.5 g) was resuspended in 5 ml ofethanol, chloroform or methylene chloride containing a) 1.5%glutaraldehyde, b) 2.0% diphthaloyl chloride or c) 5.0% formaldehyde.The microspheres were stirred for varying times from 10 minutes to 3hours. The microspheres were removed by filtration and washed thoroughlyin the original organic buffer containing 5% ethanolamine, in order toremove excess cross-linking agent. Finally the microspheres were washedin organic solvent and vacuum dried to remove any residual solvents. Theextent of insolubilisation may be varied from 5-100% by this methodresulting in the extension of in vitro echogenic half-life from 1-2minutes to in excess of one hour.

GENERAL PREPARATIVE EXAMPLE 7

The two independent steps of microsphere formation and insolubilisationof the shell may be combined in a single process. In this example, theformation of the microspheres and the insolubilisation of the polymericmaterial are achieved simultaneously during the spray drying process.

A solution of rHA was fed by peristaltic pump to a small reactionchamber, with a separate feed line supplying a 5% solution of a suitablecrosslinking agent, eg glutaraldehyde, diphthaloyl chloride orformaldehyde. The residence time in the reaction chamber was such thatinitial adduct formation between the crosslinking agent and the proteinwas achieved, but intraprotein crosslinking was prevented. The reactionvessel outlet was fed directly to the two fluid nozzle atomisers mountedin a specially adapted spray drying unit, capable of handling volatilesolvents. The conditions of spray drying were as outlined in Example 1.The microspheres were incubated dry at room temperature to allowintraprotein crosslinks to form and then suspended in ethanol containing5% ethanolamine to quench any remaining crosslinking agent. Thoroughwashing of the microspheres was performed and finally the microsphereswere vacuum dried to remove residual solvent.

GENERAL PREPARATIVE EXAMPLE 8 Assay of Free Monomeric rHA inMicrospheres

A 1 ml volume of ethanol was added to 100 mg of microspheres in a 20 mlglass bottle and sonicated for 30 seconds. To this suspension 19 ml ofH₂O were added.

The mixture was centrifuged in a bench-top microfuge (Gilson) for 20seconds and the clear fraction assayed. The assay was performed byloading 50 ml of the fraction automatically onto a Shimadzu LC6A HPLCand chromatographing on a TSK gel permeation column at a flow rate of 1ml minute⁻¹ using sodium phosphate buffer (pH 7.0).

The peak heights representing the rHA monomer were recorded and used todetermine the concentration of monomer using a standard curve between 1and 10 mgml⁻¹ monomeric rHA.

The %-free monomeric rHA was calculated by measuring the monomerconcentration in the fixed microspheres and representing this figure asa percentage of the monomer concentration of the unfixed microspheres.The results are given in FIG. 2.

Heating of the spray dried microspheres in an oven (as described inExample 1) results in a decrease in the amount of monomer that can bedetected (see FIG. 2). This decrease in detectable monomeric rHA is dueto the denaturation and crosslinking of monomeric rHA into insolublepolymers that cannot be assayed by the aforementioned HPLC method.

Using the HPLC method to assess rHA monitor levels, it is clear fromFIG. 2 that after 15 minutes incubation there is no free monomeric rHApresent in the rHA microspheres. However it is still possible to furthercrosslink the rHA microspheres by heating for longer periods.

This prolonged heating results in an increased level of microspherecrosslinking which in turn produces microspheres of increasing strengthwhich are correspondingly more resistant to pressure.

By careful control of temperature and time of incubation, it is possibleto produce microspheres with a controlled range of crosslinking (andhence pressure resistivity and biodegradation rate).

GENERAL PREPARATIVE EXAMPLE 9 Effects of Incubation Time at 175° C. onthe Pressure Resistivity of rHA Microspheres

A batch of rHA microspheres from the initial spray-drying step of theprocess was divided into 5 g aliquots and baked at 175° C. for varyinglengths of time as shown in FIG. 3.

Following heat fixation the amount of free monomer was determined asdescribed in Example 8. For each of the incubations shown, there was nomonomeric rHA detectable.

The heat-fixed microspheres were disaggregated using a Fritschcentrifugal mill (as described above) and intact, air-containingmicrospheres recovered by the aforementioned flotation technique. Therecovered microspheres were suspended in H₂O containing Pluronic F68 (1mgml⁻¹) at a concentration of 0.5×10⁸ capsules ml⁻¹.

The resuspended, air-containing microspheres were subjected to increasedatmospheric pressure by applying pressure with a 50 ml syringe whilstcontaining this suspension in a closed container (25 ml polystyrenecontainer).

For each of the pressures assessed, the individual microspheresuspension was pressurised to the selected pressure and maintained atthis pressure for 5 seconds before releasing the pressure. For eachsuspension analysed the pressure increase was performed 3 times. Thepressure in the closed container was assessed by an RS hand-heldmanometer.

Following pressurisation the microsphere suspensions were assessed bylight microscopy and image analysis and the % air-containing tonon-air-containing microspheres assessed. This analysis is performedsince only the air-containing microspheres are functional in enhancingultrasound echocontrast.

As can be seen in FIG. 3, microspheres that are fixed for 60 minutes at175° C., as described in Example 1, are stable at all of the pressuresto which they were subjected in this experiment.

By careful control of the length of incubation at this particulartemperature (175° C.) it is possible to produce batches of microsphereswith different degrees of crosslinking which in turn are resistant tovarying degrees of pressure increase.

Using this careful control of crosslinking by adjusting the length andtemperature of incubation it is possible to produce batches ofair-containing microspheres that are specifically designed to withstanda designated pressure increase.

The temperature used to crosslink the microspheres can vary infinitely,as can the length of incubation time.

GENERAL PREPARATIVE EXAMPLE 10 Microsphere Classification

An advantage of the process of the invention is that it enables themedian size and size distribution of the microspheres to be controlled.However, one can further select desired sizes if one wishes, for exampleby flotation. In a homogeneous dispersion of microspheres, largerparticles will rise to the surface faster than smaller particles due tothe lower density (more encapsulated air) of the larger particles.Hence, by allowing the dispersion to stand, the particle sizedistribution will change at any level of the solution with respect totime.

Microspheres were dispersed in 2000 ml of aqueous solution containing 6%w/v sodium chloride and 0.1% w/v Pluronic F68 in a glass bottle giving aliquid column of approximately 165 mm. A sampling tube was placed 50 mmbelow the upper liquid surface to enable removal of samples at timedintervals.

By altering the standing time and sodium chloride concentration, it waspossible to produce a variety of particle size distributions andclassify microspheres down to 2 μm.

Other wet techniques for classification include hydrodynamicchromatography and field flow fractionation. ‘Dry’ techniques using theprinciples of elutriation and cross flow separation are commerciallyavailable in the form of the Microsplit (British Rem.), Zig-zag (Alpine)and Turbo (Nissuin) classifiers. The elbow jet classifier produced byNitettsu Mining Co uses a different principle (the Coanda Effect) whichcould also achieve good results for the classification of microspheres.

SPECIFIC EXAMPLE 1

A solution of human albumin (5% w/v) is spray-dried at an inlettemperature of 220° C. and an air pressure of 1.5 bar as in GeneralPreparation Example 1. The resulting particles are heat fixed for aperiod of 20 minutes at 175° C. in an air oven. The samples aredeagglomerated by milling with mannitol and the particles areresuspended in a solution of 10 mg/ml mannitol and 0.06 mg/ml pluronicF68. The intact particles are creamed off and the microsphere suspensionis freeze-dried.

Particles predominantly of 10-20 μm are produced which contain air andare substantially pressure resistant.

SPECIFIC EXAMPLE 2

Polylysine at a concentration of 5% w/v was resuspended with themicrospheres of General Preparative Example 2 (100×10⁶ particles/ml) andincubated overnight at 20° C. Mannitol and Pluronic F68 were added atthe concentration described in Specific Example 1 and the suspension wassubsequently flash frozen and freeze dried.

SPECIFIC EXAMPLE 3

Hyaluronic acid at a concentration of 5% w/v was incubated overnightwith resuspended microspheres prepared as in General Preparative Example1 at 20° C. (100×10⁶ microspheres/ml). Mannitol and Pluronic F68 wereadded to a concentration of 10 and 0.06 mg/ml respectively and thesuspension then flash frozen and freeze dried.

SPECIFIC EXAMPLE 4

Microspheres according to General Preparative Example 3 were resuspendedin a solution of DMF (Dimethylformamide) at a concentration of 100×10⁶particles/ml. Acetic anhydride was added to give a final acid anhydrideconcentration of 100 mg/ml. The microsphere mixture was incubated at 20°C. for 1 hour then diluted with water and filtered and washed withexcess water over a 1 hour period. The microspheres were formulated inMannitol and Pluronic F68 as described above. This method impartsnegative charges.

SPECIFIC EXAMPLE 5

Microspheres according to General Preparative Example 1 were resuspendedin an aqueous solution at a concentration of 100×10⁶ particles/ml. Anaqueous solution of carbodiimide was added to the microsphere suspensionto give a final concentration of 100 mg/ml. After incubation at 16 hoursat 20° C., excess reagent was quenched by the addition of glycine to anequivalent concentration and further incubation for 16 hours at 20° C.The microspheres were washed with water then formulated as describedabove. This procedure eliminates negative charges.

SPECIFIC EXAMPLE 6

Microcapsules of general preparative method 2 were formulated withpolaxamer 407 and mannitol at a concentration of 0.1 and 10 mg/mlrespectively. The suspension was flash frozen and freeze dried asdescribed in the earlier examples.

SPECIFIC EXAMPLE 7

Poly-L-lysine (15-25 kDa) was added to the rHA feedstock (20% w/v) to afinal concentration of 0.5% w/v prior to spray drying. The method ofgeneral example 2 was followed to yield microcapsules with increasedpositive charge upon the shell.

SPECIFIC EXAMPLE 8

Poly-L-glutamate (15-30 kDa) was added to the rHA feddstock (20% w/v) toa final concentration of 0.5% w/v prior to spray drying. The method ofgeneral preparative example 2 was followed to yield microcapsules withincreased negative charge upon the shell.

SPECIFIC EXAMPLE 9

Microspheres of Specific Example 1 may be used in an in vivo analysis toestablish the feasibility of delineating perfusion territories in themyocardium of a pig heart.

A 25 kg Yorkshire swine is anaesthetised and fully ventilated accordingto the methodology outlined in Ten Cate et al (1992) CardiovascularResearch 26, 32-39. A 5 French catheter is inserted via the femoralartery, ascending aorta and aortic root into the left ventricle.Injection of 4 million microcapsules of Specific Example 1 is made and 2dimensional transthoracic echocardiography in the short axis plan usinga Hewlett Packard sono's 1000, equipped with a 3.5 MHz transducer, isused to assess regional perfusion. Intense opacification of themyocardium was observed (see FIG. 5), showing that no redistribution ofhollow microcapsules occurred over the 2 hour period. Subsequentinjections of microcapsules into the left ventricle resulted insequential dose-dependent brightening of the myocardium. Haemodynamicparameters were monitored and showed no adverse effect of injection ofthese low levels of microcapsules.

SPECIFIC EXAMPLE 10

Microcapsules of Specific Example 6 were injected into the ear vein of amildly sedated New Zealand rabbit (4.5 kg) at a concentration of 300million particles/ml. Femoral artery Doppler signals were assessed usingan Interspect 7000 model equipped with a 10 MHz transducer. Baselinesignals prior to contrast injection were obtained to enable comparisonof Doppler signals before and after contrast injection. Once baselinesignals were obtained, the instrument's time intensity gain controlswere not altered.

Following contrast injection, visible prolonged Doppler enhancement ofthe myocardium was obtained, lasting for several beats or severalminutes depending upon the dose size of contrast agent administered.

The T½ was determined by videodensitometry of the spectral Dopplersignals as follows. The gain settings were adjusted to give barelyvisible signals before contrast injection. As the contrast entered thefemoral artery the signal increased, peaked and then decayed.Videodensitometry was performed on the individual peaks of flow and atime intensity curve plotted. The T½ was calculated as the time takenfor the contrast effect to diminish to half its peak value.Videodensitometry of spectral Doppler signals revealed a reproduciblecontrast effect following intravenous injection of the microcapsuleswhich was significantly prolonged over the signals produced bymicrocapsules formulated according to PCT/GB92/00643.

1. A process of preparing microcapsules, the process comprisingspray-drying a solution or dispersion of at least one wall-formingmaterial in a liquid carrier into a gas in order to obtain gas orvapor-filled microcapsules by evaporation of said liquid carrier,wherein the solution or dispersion incorporates hyaluronic acid.
 2. Aprocess according to claim 1 wherein at least 90% of the microcapsulesare 1.0 to 8.0 μm in diameter.
 3. Hollow microcapsules predominantly of1.0-10.0 μm in diameter wherein at least 10% of the microcapsules, whensuspended in water, are capable of surviving a 0.25 s application of apressure of 2.66×10⁴ Pa without bursting, collapsing or filling withwater, and wherein the microcapsules comprise hyaluronic acid.
 4. Hollowmicrocapsules in which more than 30% of the microcapsules have adiameter within a 2 μm range and at least 90% have a diameter within therange 1.0-8.0 μm, wherein microcapsules comprise hyaluronic acid. 5.Hollow microcapsules in which the interquartile range of diameters is 2μm or less, the median diameter is between 2.0 μm and 8.0 μm inclusive,and the microcapsules comprise hyaluronic acid.
 6. Microcapsules ofwhich at least 90% are 1.0 to 8.0 μm in diameter, the microcapsulescomprising hyaluronic acid.
 7. A sterile, pyrogen-free preparationcomprising microcapsules according to any one of claims 3 to
 6. 8. Amethod of generating an image for subsequent inspection, comprising (a)injecting into the body of a mammal microcapsules according to any oneof claims 3 to 6, (b) subjecting the mammal or part thereof to suitableultrasonic radiation and (c) detecting ultrasonic radiation reflected,transmitted, resonated or frequency modulated by the said microcapsules.9. A sterile, pyrogen-free preparation comprising microcapsules, whereinsaid microcapsules are made according to the process of claim 1.